Bipolar spindle assembly is required for the correct segregation of duplicated chromosomes during cell division. While spindle assembly has been studied for many years, the mechanisms governing it remain unclear. The general goal of the project is to elucidate the principles that underlie spindle assembly and function, as well as to identify and study the roles of individual proteins. In the long term, this approach will allow the reconstitution of spindle assembly from purified components. Since uncontrolled cell division is at the heart of the cancer problem, a molecular understanding of spindle assembly and function could lead to new approaches for cancer therapy. To study spindle assembly mechanisms we use assays based on cytoplasmic extracts prepared from eggs of the frog Xenopus laevis that can recapitulate mitotic spindle assembly and anaphase chromosome segregation in vitro. The key role of chromatin in this system is illustrated by the ability of plasmid DNA-coated beads to induce the formation of bipolar spindles in the absence of centrosomes and kinetochores. However, anaphase chromosome segregation requires kinetochore function, which can be studied using sperm chromosomes. Our specific aims focus on elucidating fundamental aspects of chromosome function and microtubule organization during mitosis. The general strategies take advantage of the open nature of extracts, which allows specific inactivation of individual components, biochemical analysis of protein activities and interactions, and time-lapse video microscopy to study chromosome and microtubule morphogenesis at high resolution. Our aims are: (1) To elucidate the role of higher order chromatin architecture in chromosome functions by interfering with the major condensation machinery, the condensin complex. (2) To study the roles of the mitotic chromosome-associated kinases Plxl and Aurora B in regulating spindle assembly and function, and identify their substrates. (3) To characterize the centromeric DNA and protein components of the Xenopus kinetochore, and elucidate their functions and assembly mechanisms.